Fibroblast growth factor 2 enhances BMSC stemness through ITGA2-dependent PI3K/AKT pathway activation.

Bone marrow-derived mesenchymal stem cells (BMSC) are promising cellular reservoirs for treating degenerative diseases, tissue injuries, and immune system disorders. However, the stemness of BMSCs tends to decrease during in vitro cultivation, thereby restricting their efficacy in clinical applications. Consequently, investigating strategies that bolster the preservation of BMSC stemness and maximize therapeutic potential is necessary. Transcriptomic and single-cell sequencing methodologies were used to perform a comprehensive examination of BMSCs with the objective of substantiating the pivotal involvement of fibroblast growth factor 2 (FGF2) and integrin alpha 2 (ITGA2) in stemness regulation. To investigate the impact of these genes on the BMSC stemness in vitro, experimental approaches involving loss and gain of function were implemented. These approaches encompassed the modulation of FGF2 and ITGA2 expression levels via small interfering RNA and overexpression plasmids. Furthermore, we examined their influence on the proliferation and differentiation capacities of BMSCs, along with the expression of stemness markers, including octamer-binding transcription factor 4, Nanog homeobox, and sex determining region Y-box 2. Transcriptomic analyzes successfully identified FGF2 and ITGA2 as pivotal genes responsible for regulating the stemness of BMSCs. Subsequent single-cell sequencing revealed that elevated FGF2 and ITGA2 expression levels within specific stem cell subpopulations are closely associated with stemness maintenance. Moreover, additional in vitro experiments have convincingly demonstrated that FGF2 effectively enhances the BMSC stemness by upregulating ITGA2 expression, a process mediated by the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. This conclusion was supported by the observed upregulation of stemness markers following the induction of FGF2 and ITGA2. Moreover, administration of the BEZ235 pathway inhibitor resulted in the repression of stemness transcription factors, suggesting the substantial involvement of the PI3K/AKT pathway in stemness preservation facilitated by FGF2 and ITGA2. This study elucidates the involvement of FGF2 in augmenting BMSC stemness by modulating ITGA2 and activating the PI3K/AKT pathway. These findings offer valuable contributions to stem cell biology and emphasize the potential of manipulating FGF2 and ITGA2 to optimize BMSCs for therapeutic purposes.

Melatonin rescues the mitochondrial function of bone marrow-derived mesenchymal stem cells and improves the repair of osteoporotic bone defect in ovariectomized rats.

Osteoporotic bone defects, a severe complication of osteoporosis, are distinguished by a delayed bone healing process and poor repair quality. While bone marrow-derived mesenchymal stem cells (BMMSCs) are the primary origin of bone-forming osteoblasts, their mitochondrial function is impaired, leading to inadequate bone regeneration in osteoporotic patients. Melatonin is well-known for its antioxidant properties and regulation on bone metabolism. The present study postulated that melatonin has the potential to enhance the repair of osteoporotic bone defects by restoring the mitochondrial function of BMMSCs. In vitro administration of melatonin at varying concentrations (0.01, 1, and 100 µM) demonstrated a significant dose-dependent improvement in the mitochondrial function of BMMSCs obtained from ovariectomized rats (OVX-BMMSCs), as indicated by an elevation in mitochondrial membrane potential, adenosine triphosphate synthesis and expression of mitochondrial respiratory chain factors. Melatonin reduced the level of mitochondrial superoxide by activating the silent information regulator type 1 (SIRT1) and its downstream antioxidant enzymes, particularly superoxide dismutase 2 (SOD2). The protective effects of melatonin were found to be nullified upon silencing of Sirt1 or Sod2, underscoring the crucial role of the SIRT1-SOD2 axis in the melatonin-induced enhancement of mitochondrial energy metabolism in OVX-BMMSCs. To achieve a sustained and localized release of melatonin, silk fibroin scaffolds loaded with melatonin (SF@MT) were fabricated. The study involved the surgical creation of bilateral femur defects in OVX rats, followed by the implantation of SF@MT scaffolds. The results indicated that the application of melatonin partially restored the mitochondrial energy metabolism and osteogenic differentiation of OVX-BMMSCs by reinstating mitochondrial redox homeostasis. These findings suggest that the localized administration of melatonin through bone implants holds potential as a therapeutic approach for addressing osteoporotic bone defects.

Vitamin D supplementation affects bone marrow-derived mesenchymal stem cells differentiation into insulin-producing cells.

Background this study was conducted to assess the effects of vitamin D on differentiation of bone marrow- derived mesenchymal stem cells (BM-MSCs) into insulin producing cells (IPCs). Method BM-MSCs were isolated from femur and tibia of rats and incubated in low (LG) or high glucose (HG) (5mM or 25mM), or high glucose DMEM media supplemented with vitamin D (0.2nM) (HGD) for 14 days. Cells viability was analysis by MTT assay. Differentiation of SCs was confirmed using measuring genes expression level of pdx1 and insulin, and insulin secretion, glucose stimulated insulin secretion, and insulin content by ELISA method. Results Cell viability was significantly higher in HGD than LG (p < 0.05) in day 3, also, in HG and HGD than LG (p < 0.001), and HGD vs. HG (p < 0.001) in day 7. Pdx1 and insulin level was markedly higher in HGD than LG (p < 0.05 and p < 0.01). pdx1 expression was markedly higher in HGD (p < 0.05) than LG, also insulin expression the HG (p < 0.05), and HGD (p < 0.01) groups compared to the LG group. Insulin release at 5mM glucose was notably higher in the HGD group compared to LG (p < 0.05), and at 25mM glucose, both HG and HGD showed significant increases vs. LG (p < 0.05 and p < 0.01, respectively). Insulin content was significantly higher in both 5mM and 25mM glucose for HG and HGD vs. LG (p < 0.01 and p < 0.001, respectively). In conclusion, treatment BM-MSCs with vitamin D could increase their differentiation into IPCs and it can be considered as a potential supplementary agent in enhancing differentiation SCs into insulin generating cells.

Vimentin protein is a factor for decreasing breast cancer cell proliferation co-culture with human bone marrow-derived mesenchymal stem cells pre-treated with thiazolidinedione solutions.

BACKGROUND: Our previous study investigated the levels of soluble growth factors in the conditioned media of bone marrow-derived mesenchymal stem cells (BMSCs) pre-treated with thiazolidinedione solutions. The present study aimed to investigate the complex intracellular proteins extracted from BMSCs pre-treated with pioglitazone and/or rosiglitazone using proteomics. METHODS: The proliferative effect of the identified protein on MCF-7 cells that interacted non-adhesively with BMSCs pre-treated with pioglitazone and/or rosiglitazone was evaluated using cell culture inserts and conditioned media. The mRNA expression of proliferation and lipid accumulation markers was also evaluated in the interacted MCF-7 cells by reverse transcription-quantitative PCR. Finally, the correlation between the identified protein and fibroblast growth factor 4 (FGF-4) protein in the conditioned media of the pre-treated BMSCs was evaluated by ELISA. RESULTS: The present study identified vimentin as the specific protein among the complex intracellular proteins that likely plays a role in MCF-7 cell proliferation when the breast cancer cells interacted non-adhesively with BMSCs pre-treated with a combination of pioglitazone and rosiglitazone. The inhibition of this protein promoted the proliferation of MCF-7 cells when the breast cancer cells interacted with pre-treated BMSCs. Gene expression analysis indicated that pre-treatment of BMSCs with a combination of pioglitazone and rosiglitazone decreased the mRNA expression of Ki67 and proliferating cell nuclear antigen in MCF-7 cells. The pre-treatment did not induce mRNA expression of PPARγ, which is a sign of lipid accumulation. The level of vimentin protein was also associated with the FGF-4 protein expression level in the conditioned media of the pre-treated BMSCs. Bioinformatics analysis revealed that vimentin regulated the expression of FGF-4 through its interaction with SRY-box 2 and POU class 5 homeobox 1. CONCLUSIONS: The present study identified a novel intracellular protein that may represent the promising target in pre-treated BMSCs to decrease the proliferation of breast cancer MCF-7 cells for human health and wellness.

Osteogenic potentials in canine mesenchymal stem cells: unraveling the efficacy of polycaprolactone/hydroxyapatite scaffolds in veterinary bone regeneration.

BACKGROUND: The integration of stem cells, signaling molecules, and biomaterial scaffolds is fundamental for the successful engineering of functional bone tissue. Currently, the development of composite scaffolds has emerged as an attractive approach to meet the criteria of ideal scaffolds utilized in bone tissue engineering (BTE) for facilitating bone regeneration in bone defects. Recently, the incorporation of polycaprolactone (PCL) with hydroxyapatite (HA) has been developed as one of the suitable substitutes for BTE applications owing to their promising osteogenic properties. In this study, a three-dimensional (3D) scaffold composed of PCL integrated with HA (PCL/HA) was prepared and assessed for its ability to support osteogenesis in vitro. Furthermore, this scaffold was evaluated explicitly for its efficacy in promoting the proliferation and osteogenic differentiation of canine bone marrow-derived mesenchymal stem cells (cBM-MSCs) to fill the knowledge gap regarding the use of composite scaffolds for BTE in the veterinary orthopedics field. RESULTS: Our findings indicate that the PCL/HA scaffolds substantially supported the proliferation of cBM-MSCs. Notably, the group subjected to osteogenic induction exhibited a markedly upregulated expression of the osteogenic gene osterix (OSX) compared to the control group. Additionally, the construction of 3D scaffold constructs with differentiated cells and an extracellular matrix (ECM) was successfully imaged using scanning electron microscopy. Elemental analysis using a scanning electron microscope coupled with energy-dispersive X-ray spectroscopy confirmed that these constructs possessed the mineral content of bone-like compositions, particularly the presence of calcium and phosphorus. CONCLUSIONS: This research highlights the synergistic potential of PCL/HA scaffolds in concert with cBM-MSCs, presenting a multidisciplinary approach to scaffold fabrication that effectively regulates cell proliferation and osteogenic differentiation. Future in vivo studies focusing on the repair and regeneration of bone defects are warranted to further explore the regenerative capacity of these constructs, with the ultimate goal of assessing their potential in veterinary clinical applications.

The Impact of Centrifugal Force on Isolation of Bone Marrow Mononuclear Cells Using Density Gradient Centrifugation.

BACKGROUND: Bone marrow mononuclear cells (BMMNCs) have great potential in bone regenerative therapy. The main method used today to obtain BMMNCs is Ficoll density gradient centrifugation. However, the centrifugal force for this isolation method is still suboptimal. OBJECTIVES: To determine the optimal centrifugal force in Ficoll density gradient centrifugation of bone marrow (BM) to achieve high stem/progenitor cell content BMMNCs for regenerative therapy. METHODS: BM was aspirated from nine minipigs and divided into three groups according to different centrifugal forces (200 g, 300 g and 400 g). Immediately after BMMNCs were obtained from each group by Ficoll density gradient centrifugation, residual red blood cell (RBC) level, nucleated cell counting, viability and flow cytometric analyses of apoptosis and reactive oxygen species (ROS) generation were measured. The phenotypic CD90 and colony formation analyses of BMMNCs of each group were performed as well. Bone marrow-derived mesenchymal stem cells (BMSCs) were harvested at passage 2, then morphology, cell phenotype, proliferation, adipogenic, chondrogenic and osteogenic lineage differentiation potential of BMSCs from each group were compared. RESULTS: The 300 g centrifugal force was able to isolate BMMNCs from BM with the same efficiency as 400 g and provided significantly higher yields of CD90+ BMSCs and fibroblastic colony-forming units of BMSC (CFU-f(BMSC)), which is more crucial for the regenerative efficacy of BMMNCs. Meanwhile, 200 g hosted the most RBC contamination and minimum CFU-f (BMSC) yield, which will be disadvantageous for BMMNC-based cell therapy. As for in vitro cultured BMSCs which were isolated from BMMNCs by different centrifugal forces, no significant differences were found on morphology, cell proliferation rate, phenotypic marker, adipogenic, chondrogenic and osteogenic differentiation potential. CONCLUSIONS: 300 g may be the optimal centrifugal force when using Ficoll density gradient centrifugation to isolate BMMNCs for bone regenerative therapy. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Hypoxic preconditioned mesenchymal stem cells ameliorate rat brain injury after cardiopulmonary resuscitation by suppressing neuronal pyroptosis.

Cardiac arrest (CA) can result in cerebral ischaemia-reperfusion injury and poor neurological outcomes. While bone marrow-derived mesenchymal stem cells (BMSCs) have been shown to have protective effects in brain ischaemic disease, their efficacy can be reduced by the poor oxygen environment. In this study, we investigated the neuroprotective effects of hypoxic preconditioned BMSCs (HP-BMSCs) and normoxic BMSCs (N-BMSCs) in a cardiac arrest rat model by examining their ability to ameliorate cell pyroptosis. The mechanism underlying the process was also explored. Cardiac arrest was induced in rats for 8 min and surviving rats received 1 × 106 normoxic/hypoxic BMSCs or PBS via intracerebroventricular (ICV) transplantation. Neurological function of rats was evaluated using neurological deficit scores (NDSs) and examined for brain pathology. Serum S100B and neuron-specific enolase (NSE) levels and cortical proinflammatory cytokines were measured to evaluate brain injury. Pyroptosis-related proteins in the cortex after cardiopulmonary resuscitation (CPR) were measured using western blotting and immunofluorescent staining. Transplanted BMSCs were tracked using bioluminescence imaging. Results showed significantly better neurological function and neuropathological damage after transplantation with HP-BMSCs. In addition, HP-BMSCs reduced levels of pyroptosis-related proteins in the rat cortex after CPR and significantly reduced levels of biomarkers for brain injury. Mechanistically, HP-BMSCs alleviated brain injury by reducing the expressions of HMGB1, TLR4, NF-κB p65, p38 MAPK and JNK in the cortex. Our study demonstrated that hypoxic preconditioning could enhance the efficacy of BMSCs in alleviating post-resuscitation cortical pyroptosis. This effect may be related to the regulation of the HMGB1/TLR4/NF-κB, MAPK signalling pathways.

BMP6 participates in the pathogenesis of adolescent idiopathic scoliosis by regulating osteopenia.

Adolescent idiopathic scoliosis (AIS) is a complex disease characterized by three-dimensional structural deformities of the spine. Its pathogenesis is associated with osteopenia. Bone-marrow-derived mesenchymal stem cells (BMSCs) play an important role in bone metabolism. We detected 1919 differentially expressed mRNAs and 744 differentially expressed lncRNAs in BMSCs from seven patients with AIS and five patients without AIS via high-throughput sequencing. Multiple analyses identified bone morphogenetic protein-6 (BMP6) as a hub gene that regulates the abnormal osteogenic differentiation of BMSCs in AIS. BMP6 expression was found to be decreased in AIS and its knockdown in human BMSCs significantly altered the degree of osteogenic differentiation. Additionally, CAP1-217 has been shown to be a potential upstream regulatory molecule of BMP6. We showed that CAP1-217 knockdown downregulated the expression of BMP6 and the osteogenic differentiation of BMSCs. Simultaneously, knockout of BMP6 in zebrafish embryos significantly increased the deformity rate. The findings of this study suggest that BMP6 is a key gene that regulates the abnormal osteogenic differentiation of BMSCs in AIS via the CAP1-217/BMP6/RUNX2 axis.

The ameliorating effects of mesenchymal stem cells compared to α-tocopherol on apoptosis and autophagy in streptozotocin-induced diabetic rats: Implication of PI3K/Akt signaling pathway and entero-insular axis.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are considered a novel regenerative therapy that holds much potential. This study aimed to examine and compare the ameliorative effects of BM-MSCs compared to α-tocopherol (α-Toc) on apoptosis, autophagy, and ß-cell function in a rat model of streptozotocin (STZ)-induced diabetes and further analyzed the implications and interrelations of the entero-insular axis, and type I phosphoinositide 3-kinase (PI3K)/Akt signaling. Forty adult male albino rats were categorized into four groups (n = 10, in each): control group, STZ-induced diabetic group (single i.p. injection of STZ 45 mg/kg), diabetic and treated with BM-MSCs injection, diabetic and treatment with α-Toc p.o. The serum glucose, insulin, nitric oxide (NO), and catalase (CAT) were measured. Histopathological examination of the pancreas, the expression levels of insulin, CD44, caspase-3, autophagy markers, P13K/Akt, and pancreas/duodenum homeobox protein 1, in pancreatic tissue, and glucose-dependent insulinotropic polypeptide (GIP) in the duodenum were detected by hematoxylin and eosin staining, immunofluorescence labeling, and by quantitative real-time polymerase chain reaction. The diabetic rats showed reduced insulin, hyperglycemia, nitrosative stress (NO, CAT), augmented apoptosis (caspase 3), impaired autophagy (p62/SQSTM1, LC3), downregulated PI3K/Akt pathway and increased GIP expression, and degeneration of pancreatic islets. Treatment with either BM-MSCs or α-Toc suppressed the nitrosative stress, reduced apoptosis, recovered autophagy, upregulated PI3K/Akt pathway, and subsequently increased insulin levels, decreased blood glucose, and downregulated GIP expression with partial restoration of pancreatic islets. Based on our findings, the cytoprotective effects of BM-MSCs and α-Toc in type 1-induced diabetes appeared to be related to repaired autophagy and recovered PI3K/Akt signaling. Moreover, we reported their novel effects on reversing intestinal GIP expression level. The effect of BM-MSCs was notably superior to that of α-Toc.

Effect of Atorvastatin on Angiogenesis-Related Genes VEGF-A, HGF and IGF-1 and the Modulation of PI3K/AKT/mTOR Transcripts in Bone-Marrow-Derived Mesenchymal Stem Cells.

Stem cell transplantation represents a unique therapeutic tool in tissue engineering and regenerative medicine. However, it was shown that the post-injection survival of stem cells is poor, warranting a more comprehensive understanding of activated regenerative pathways. Numerous studies indicate that statins improve the therapeutic efficacy of stem cells in regenerative medicine. In the present study, we investigated the effect of the most widely prescribed statin, atorvastatin, on the characteristics and properties of bone-marrow-derived mesenchymal stem cells (BM-MSCs) cultured in vitro. We found that atorvastatin did not decrease the viability of BM-MSCs, nor did it change the expression of MSC cell surface markers. Atorvastatin upregulated the mRNA expression levels of VEGF-A and HGF, whereas the mRNA expression level of IGF-1 was decreased. In addition, the PI3K/AKT signaling pathway was modulated by atorvastatin as indicated by the high mRNA expression levels of PI3K and AKT. Moreover, our data revealed the upregulation of mTOR mRNA levels; however, no change was observed in the BAX and BCL-2 transcripts. We propose that atorvastatin benefits BM-MSC treatment due to its ability to upregulate angiogenesis-related genes expression and transcripts of the PI3K/AKT/mTOR pathway.

Inokosterone activates the BMP2 to promote the osteogenic differentiation of bone marrow mesenchymal stem cells and improve bone loss in ovariectomized rats.

Evidence suggests that enhancing the osteogenic ability of bone marrow-derived mesenchymal stem cells (BMSCs) may be beneficial in the fight against osteoporosis (OP) effects. Inokosterone (IS) is a major active constituent of Achyranthis bidentatae radix (ABR), which stimulates osteogenic differentiation of mouse embryonic osteoblasts. This study aims to investigate effect of IS on OP using osteogenic differentiated BMSCs and ovariectomy (OVX)-induced OP rats. The BMSCs were treated with 50, 100, or 200 mg/L IS and OP rats were given 2 or 4 mg/kg of IS by gavage. Cell viability, the osteogenic differentiation marker protein expression level, and mineralization were observed. This study proved that IS improved cell viability, osteogenic differentiation, and cellular mineralization in BMSCs and raised expression levels of bone morphogenetic protein-2 (BMP2), Smad1, runt-related transcription factor 2 (RUNX2), collagen I, ALP, and OCN. By BMP2 knockdown/overexpression, this study also proved the BMP2 signaling pathway activation is a potential biological mechanism of IS to improve osteogenic differentiation and mineralization in osteogenic differentiated BMSCs. In OVX-induced OP rats, IS was observed to antagonize bone loss, improve osteogenic differentiation marker protein expression levels, and activate BMP-2, smad1, and RUNX2. These findings provide scientific support for further investigation of the biological mechanisms of IS in ameliorating OP.

Bone marrow-derived mesenchymal stem cells transplantation attenuates renal fibrosis following acute kidney injury by repairing the peritubular capillaries.

After the severe initial insults of acute kidney injury, progressive kidney tubulointerstitial fibrosis may occur, the peritubular capillary (PTC) rarefaction plays a key role in the disease progression. However, the mechanisms of PTC damage were not fully understood and potential therapeutic interventions were not explored. Previous studies of our research team and others in this field suggested that bone marrow-derived mesenchymal stem cells (BMSCs) transplanted into the AKI rat model may preserve the kidney function and pathological changes. In the current study, with the ischemia/reperfusion AKI rat model, we revealed that BMSCs transplantation attenuated the renal function decrease in the AKI model through preserving the peritubular capillaries (PTCs) function. The density of PTCs is maintained by BMSCs transplantation in the AKI model, detachment and relocation of pericytes in the PTCs diminished. Then we established that BMSCs transplantation may attenuate the renal fibrosis and preserve the kidney function after AKI by repairing the PTCs. Improving the vitality of pericytes, suppressing the detachment and trans-differentiation of pericytes, directly differentiation of BMSCs into pericytes by BMSCs transplantation all participate in the PTC repair. Through these processes, BMSCs rescued the microvascular damage and improved the density of PTCs. As a result, a preliminary conclusion can be reached that BMSCs transplantation can be an effective therapy for delaying renal fibrosis after AKI.

Extracellular vesicles-encapsulated microRNA-29b-3p from bone marrow-derived mesenchymal stem cells promotes fracture healing via modulation of the PTEN/PI3K/AKT axis.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are well-established as vital regulators of fracture healing, whereas angiogenesis is one of the critical processes during the course of bone healing. Accordingly, the current study sought to determine the functions of microRNA (miR)-29b-3p from BM-MSCs-derived extracellular vesicles (EVs) on the angiogenesis of fracture healing via the PTEN/PI3K/AKT axis. Firstly, BM-MSCs-EVs were extracted and identified. The lentiviral protocol was adopted to construct miR-29b-3pKD-BMSCs or miR-negative control-BMSCs, which were then co-cultured with human umbilical vein endothelial cells (HUVECs) in vitro to determine the roles of EVs-encapsulated miR-29b-3p on the proliferation, migration, and angiogenesis of HUVECs in vitro with the help of a CCK-8 assay, scratch test, and tube formation assay. Subsequent database prediction, luciferase activity assay, RT-qPCR, and Western blot assay findings identified the downstream target gene of miR-29b-3p, PTEN, and a signaling pathway, PI3K/AKT. Furthermore, the application of si-PTEN attenuated the effects induced by miR-29b-3pKD-EVs. Finally, a mouse model of femoral fracture was established with a locally instilled injection of equal volumes of BM-MSCs-EVs and miR-29b-3pKD-BM-MSCs-EVs. Notably, the mice treated with BMSC-EVs presented with enhanced neovascularization at the fracture site, in addition to increased bone volume (BV), BV/tissue volume, and mean bone mineral density; whereas miR-29b-3pKD-BMSCs-EVs-treated mice exhibited decreased vessel density with poor fracture healing capacity. Collectively, our findings elicited that BM-MSCs-EVs carrying miR-29b-3p were endocytosed by HUVECs, which consequently suppressed the PTEN expression and activated the PI3K/AKT pathway, thereby promoting HUVEC proliferation, migration, and angiogenesis, and ultimately facilitating fracture healing.

Ectopic expression of insulin in a type 1 diabetic rat model by injection of manipulated mesenchymal stem cells with an insulin construct driven by a glucose-sensitive promoter in the port vein.

The treatment of type 1 diabetes through islet cell transplantation is a complex process, facing challenges such as allograft rejections and a limited supply of donors. One potential solution is to utilize the liver as an alternative for natural insulin production, as hepatocytes can secrete proteins and respond to glucose levels. Recent research has shown promising results in using mesenchymal stem cells as a potential cure for diabetes. The study utilized a diabetic rat model, confirmed through blood sugar measurement. A plasmid vector was designed with specific genetic components, synthesized by biotech company, and then Inserted vector into a plasmid with resistance genes and bacterial origin. Bone marrow-derived mesenchymal stem cells (BM-MSCs) were cultured and transfected with the plasmid using Lipofectamine 3000. Polymerase chain reaction was employed to confirm successful transfection using specific primers. For the animal study, 30 male Wistar rats were divided into six groups, each comprising five rats. The control group did not receive any treatment, while the second group received MSCs via Portal Vein Injection. The third group received MSCs transfected with a specific construct via Portal Vein Injection. The fourth group was induced to develop diabetes through streptozotocin (STZ) injection, the fifth group developed diabetes and received untransfected MSCs via Portal Vein Injection, and the sixth group received MSCs transfected with the specific construct via Portal Vein Injection. To manage Pain, appropriate pain control was administered to the rats for 3 days after the surgery. Fixed liver tissues obtained from the euthanized rats were utilized for immunohistochemistry. In this study, immunohistochemical techniques were used to examine insulin expression in different groups of rats. The control groups showed high levels of insulin expression, while the diabetic groups exhibited lower expression. However, there was a significant difference between the diabetic groups treated with MSC and transgenic MSC cells. All groups had similar baseline glucose levels, but the diabetic groups showed a significant increase after STZ injection, whereas the control and MSC groups did not. Postintervention, both the control and MSC groups had similar glucose levels to the post-STZ levels. However, diabetes-induced groups experienced a significant decrease in glucose levels, with the transfected MSCs showing a greater decrease than the untransfected MSCs. The study suggested that treatment with MSCs, especially transfected ones, can effectively reduce glucose levels in rats with diabetes. In this research, rat BM-MSCs were utilized to create insulin-producing mesenchymal cells with glucose-sensitive insulin expression. The cells were transferred to the liver of diabetic rats via portal vein injection, leading to an increase in insulin expression. This study proposes a novel approach for cell therapy and delivery in the treatment of type 1 diabetes.

Evaluation of bone marrow-derived mesenchymal stem cells with eggshell membrane for full-thickness wound healing in a rabbit model.

Biomaterials capable of managing wounds should have essential features like providing a natural microenvironment for wound healing and as support material for stimulating tissue growth. Eggshell membrane (ESM) is a highly produced global waste due to increased egg consumption. The unique and fascinating properties of ESM allow their potential application in tissue regeneration. The wound healing capacity of bone marrow-derived mesenchymal stem cells (BM-MSCs), ESM, and their combination in rabbits with full-thickness skin defect (2 × 2 cm2) was evaluated. Twenty-five clinically healthy New Zealand White rabbits were divided into five groups of five animals each, with group A receiving no treatment (control group), group B receiving only fibrin glue (FG), group C receiving FG and ESM as a dressing, group D receiving FG and BM-MSCs, and group E receiving a combination of FG, ESM, and BM-MSCs. Wound healing was assessed using clinical, macroscopical, photographic, histological, histochemical, hematological, and biochemical analysis. Macroscopic examination of wounds revealed that healing was exceptional in group E, followed by groups D and C, compared to the control group. Histopathological findings revealed improved quality and a faster rate of healing in group E compared to groups A and B. In addition, healing in group B treated with topical FG alone was nearly identical to that in control group A. However, groups C and D showed improved and faster recovery than control groups A and B. The macroscopic, photographic, histological, and histochemical evaluations revealed that the combined use of BM-MSCs, ESM, and FG had superior and faster healing than the other groups.

Bone Marrow-Derived Mesenchymal Stem Cell Implants for the Treatment of Focal Chondral Defects of the Knee in Animal Models: A Systematic Review and Meta-Analysis.

Osteoarthritis remains an unfortunate long-term consequence of focal cartilage defects of the knee. Associated with functional loss and pain, it has necessitated the exploration of new therapies to regenerate cartilage before significant deterioration and subsequent joint replacement take place. Recent studies have investigated a multitude of mesenchymal stem cell (MSC) sources and polymer scaffold compositions. It is uncertain how different combinations affect the extent of integration of native and implant cartilage and the quality of new cartilage formed. Implants seeded with bone marrow-derived MSCs (BMSCs) have demonstrated promising results in restoring these defects, largely through in vitro and animal studies. A PRISMA systematic review and meta-analysis was conducted using five databases (PubMed, MEDLINE, EMBASE, Web of Science, and CINAHL) to identify studies using BMSC-seeded implants in animal models of focal cartilage defects of the knee. Quantitative results from the histological assessment of integration quality were extracted. Repair cartilage morphology and staining characteristics were also recorded. Meta-analysis demonstrated that high-quality integration was achieved, exceeding that of cell-free comparators and control groups. This was associated with repair tissue morphology and staining properties which resembled those of native cartilage. Subgroup analysis showed better integration outcomes for studies using poly-glycolic acid-based scaffolds. In conclusion, BMSC-seeded implants represent promising strategies for the advancement of focal cartilage defect repair. While a greater number of studies treating human patients is necessary to realize the full clinical potential of BMSC therapy, high-quality integration scores suggest that these implants could generate repair cartilage of substantial longevity.

HPF Modulates the Differentiation of BMSCs into HLCs and Promotes the Recovery of Acute Liver Injury in Mice.

Bone marrow-derived mesenchymal stem cells (BMSCs) can differentiate into hepatocyte-like cells (HLCs) to alleviate acute liver injury (ALI). Herpetfluorenone (HPF), as an active ingredient in the dried, mature seeds Herpetospermum caudigerum Wall, used in Tibetan medicine, has been proven to effectively alleviate ALI. Therefore, the purpose of this study was to determine whether HPF can promote the differentiation of BMSCs into HLCs and promote ALI recovery. Mouse BMSCs were isolated, and the BMSCs' differentiation into HLCs was induced by HPF and hepatocyte growth factor (HGF). Under the induction of HPF and HGF, the expression of hepatocellular specific markers and the accumulation of glycogen and lipids in the BMSCs increased, indicating that BMSCs successfully differentiated into HLCs. Then, the ALI mouse model was established, using carbon tetrachloride, followed by an intravenous injection of BMSCs. Then, only HPF was injected intraperitoneally, in order to verify the effect of HPF in vivo. In vivo imaging was used to detect the homing ability of HPF-BMSCs, and it was detected that HPF-BMSCs significantly increased the levels of serum AST, ALT and ALP in the liver of ALI mice, and alleviated liver cell necrosis, oxidative stress and liver pathology. In conclusion, HPF can promote the differentiation of BMSCs into HLCs and promote the recovery of ALI in mice.

Extracellular vesicles derived from human bone marrow mesenchymal stem cells protect rats against acute myocardial infarction-induced heart failure.

Extracellular vesicles (EVs) derived from human bone marrow mesenchymal stem cells (BMSCs) are suggested to promote angiogenesis in a rat model of acute myocardial infarction (AMI). This study aimed to explore the underlying mechanism of BMSCs-EVs in AMI-induced heart failure (HF). BMSCs were isolated and verified, and EVs were purified and identified. After establishment of AMI-induced HF models, rats were treated with BMSCs-EVs and/or overexpressing (ov)/knocking down (kd) bone morphogenetic protein 2 (BMP2). Cardiac function, myocardial histopathological changes, angiogenesis, and vascular regeneration density were measured. Levels of pro-angiogenesis factors and cardiomyocyte apoptosis were detected. The viability and angiogenesis of hypoxic human umbilical vein endothelial cells (HUVECs) were measured. After BMSCs-EV treatment, the cardiac function of HF rats was improved, myocardial fibrosis and inflammatory cell infiltration were decreased, angiogenesis was increased, and cardiomyocyte apoptosis was inhibited. BMP2 was significantly upregulated in the myocardium. Ov-BMP2-BMSCs-EVs alleviated myocardial fibrosis and inflammatory cell infiltration, and promoted angiogenesis of HF rats, and improved the activity and angiogenesis of hypoxic HUVECs, while kd-BMP2-BMSCs-EVs showed limited protection against AMI-induced HF. BMSCs-EVs deliver BMP2 to promote angiogenesis and improve cardiac function of HF rats.

Bone marrow-derived mesenchymal stem cells improve rat islet graft revascularization by upregulating ISL1.

Revascularization of the islet transplant is a crucial step that defines the success rate of patient recovery. Bone marrow-derived mesenchymal stem cells (BMSCs) have been reported to promote revascularization; however, the underlying cellular mechanism remains unclear. Moreover, our liquid chromatography-tandem mass spectrometry results showed that BMSCs could promote the expression of insulin gene enhancer binding protein-1 (ISL1) in islets. ISL1 is involved in islets proliferation and plays a potential regulatory role in the revascularization of islets. This study identifies the ISL1 protein as a potential modulator in BMSCs-mediated revascularization of islet grafts. We demonstrated that the survival rate and insulin secretion of islets were increased in the presence of BMSCs, indicating that BMSCs promote islet revascularization in a coculture system and rat diabetes model. Interestingly, we also observed that the presence of BMSCs led to an increase in ISL1 and vascular endothelial growth factor A (VEGFA) expression in both islets and the INS-1 rat insulinoma cell line. In silico protein structure modeling indicated that ISL1 is a transcription factor that has four binding sites with VEGFA mRNA. Further results showed that overexpression of ISL1 increased both the abundance of VEGFA transcripts and protein accumulation, while inhibition of ISL1 decreased the abundance of VEGFA. Using a ChIP-qPCR assay, we demonstrated that direct molecular interactions between ISL1 and VEGFA occur in INS-1 cells. Together, these findings reveal that BMSCs promote the expression of ISL1 in islets and lead to an increase in VEGFA in islet grafts. Hence, ISL1 is a potential target to induce early revascularization in islet transplantation.

Neuroprotection of Bone Marrow-Derived Mesenchymal Stem Cell-Derived Extracellular Vesicle-Enclosed miR-410 Correlates with HDAC4 Knockdown in Hypoxic-Ischemic Brain Damage.

Evidence exists reporting that miR-410 may rescue neurological deficits, neuronal injury, and neuronal apoptosis after experimental hypoxic ischemia. This study aimed to explore the mechanism by which miR-410 transferred by bone marrow-derived mesenchymal stem cell-derived extracellular vesicles (BMSC-EVs) may alleviate hypoxic-ischemic brain damage (HIBD) in newborn mice. BMSCs were isolated from total bone marrow cells of femur and tibia of newborn mice, and primary neurons were extracted from the cerebral cortex of newborn mice within 24 h of birth. EVs were extracted from BMSCs transfected with the mimic or inhibitor of miR-410. Primary neurons were subjected to hypoxia and treated with overexpression (oe)-HDAC4, small interfering RNA (siRNA)-ß-catenin, or Wnt pathway inhibitor and/or EV (miR-410 mimic) or EV (miR-410 inhibitor). A neonatal mouse HIBD model was established and treated with EVs. When BMSC-EVs were endocytosed by primary neurons, miR-410 was upregulated, neuronal viability was elevated, and apoptosis was inhibited. miR-410 in BMSC-EVs targeted HDAC4, thus increasing neuronal viability and reducing apoptosis. Conversely, overexpression of HDAC4 activated the Wnt pathway and enhanced the nuclear translocation of ß-catenin. Treatment with miR-410-containing BMSC-EVs improved learning and memory abilities of HIBD mice while attenuating apoptosis by inactivating the Wnt pathway via targeting HDAC4. Taken together, the findings suggest that miR-410 delivered by BMSC-EVs alleviates HIBD by inhibiting HDAC4-dependent Wnt pathway activation.